Bacteria that produce carbapenemases, such as the K. pneumoniae carbapenemase (blaKPC), are increasingly common multidrug resistant pathogens causing nosocomial infections and outbreaks, with high rates of morbidity and mortality worldwide. Horizontal transfer of carbapenemase-producing plasmids is believed to play a significant role in the spread of multidrug resistant K. pneumoniae and other Gram-negative bacteria. Our experiments are aimed at defining the parameters regulating the spread of carbapenem resistance. Patients can be colonized by KPC+ K. pneumoniae for extended periods of time; however, only some of these patients have evidence of blaKPC containing plasmids transferring to bacteria other than Klebsiella species based on our extensive surveillance system. Understanding any differences between in vitro potential to spread and in vivo evidence of spread will aid our attempts to prevent dissemination of the resistance phenotype. We found that investigators using in vitro models of conjugation should incorporate several parameters before drawing conclusions about a plasmids conjugative ability. Studies utilizing conjugation assays overwhelmingly use only a single assay condition. In vitro and in vivo studies of the same plasmid are often discrepant. Thus, we sought to use an even more accurate system, comparing in vitro data to in situ data, by using environmental and clinical epidemiology. Although this has been attempted previously, other studies typically use a single donor that is representative of an outbreak to a single recipient. In this study, we leverage four donor strains each with a unique molecular epidemiology, and leverage 16 patient isolate recipients not previously available for study. We generated a unique set of reagents through transformation into a plasmid-cured strain that allow for easy separation and testing of donor strain, plasmid, and recipient strain influences. Our systematic analysis showed that conjugation is heavily context-specific, and is dependent on the donor, recipient, plasmid, and the environment. The environment as a location for horizontal gene transfer has been examined and discussed, but the full extent and impact from this source of additional resistant strains is not yet known. We also sought to correlate our in vitro data to the epidemiology of blaKPC+ patients from two medical centers. We note that, surprisingly, in vitro conjugation efficiency does not correlate with observed promiscuous interspecies spread. This is in contract to conclusion of some outbreak papers that suggest horizontal transfer may be of high efficiency between different species of Enterobacteriaceae. Next, we analyzed efficient and refractory recipients by single-molecule, real-time sequencing. We used this whole genome sequence data to generate hypotheses for possible mechanisms that control conjugation, highlighting the complexity of the regulatory system for conjugation. These studies integrate epidemiology, plasmid biology, and genomics to give novel insights on clinically significant resistant Gram-negative bacteria.